Markers of acute kidney failure

ABSTRACT

The present invention relates to a method of determining the risk of acute kidney injury comprising determining the amount of a marker selected from VCAN, NRP1, CCL2, CCL19, COL3A1, GZMM or any combination thereof in a sample.

The present invention relates to a method for detection, diagnosis,prognosis, or monitoring the risk of acute kidney injury (AKI) bymeasuring a panel of biomarkers. In particular, the invention refers toa predisposition testing.

AKI is in the clinical setting described as acute renal failure (ARF) oracute tubular necrosis (ATN) and refers to the spontaneous andsignificant decrease in renal function. AKI therefore reflects theentire spectrum of ARF, recognizing that an acute decline in kidneyfunction is often secondary to an injury that causes functional orstructural changes in the kidneys. ARF is a frequent and serious problemwith a variety of adverse short- and long-term clinical consequences.Loss of function of the kidney, a vital organ, in the form of acuterenal failure represents a special hazard, in particular to olderpatients, despite modern therapies including the use of the variousforms of artificial kidney. In diagnosis and prognosis care must betaken to differentiate between functional renal insufficiency andintrinsic injury with morphologic damage.

AKI in particular in the intensive care unit is often associated withmultiple organ failure and sepsis. Furthermore, AKI is associated withhigh mortality and morbidity in humans. Patients, for instance,experience AKI in ischemic reperfusion injury, along with treatment withnephrotoxic compounds including but not limited to antibiotics oranticancer drugs, application of contrast media e.g. when performingangiography resulting in nephropathy or nephrotoxicity, or at theintensive care unit, e.g. in the context of sepsis. The annual number ofpatients receiving contrast media is more than 100 million in thedeveloped countries, and the rate of acute kidney injury ranges in apercent range, if coupled to risk factors like hypotension or diabetes.

AKI is usually categorised according to pre-renal, intrinsic andpost-renal causes.

Pre-renal (causes in the blood supply):

-   -   hypovolemia (decreased blood volume), usually from shock or        dehydration and fluid loss or excessive diuretics use.    -   hepatorenal syndrome, in which renal perfusion is compromised in        liver failure    -   vascular problems, such as atheroembolic disease and renal vein        thrombosis (which can occur as a complication of the nephrotic        syndrome)    -   infection usually sepsis, systemic inflammation due to infection    -   severe burns    -   sequestration due to pericarditis and pancreatitis    -   hypotension due to antihypertensives and vasodilators

Intrinsic (damage to the kidney itself):

-   -   toxins or medication (e.g. some NSAIDs, aminoglycoside        antibiotics, iodinated contrast, lithium, phosphate nephropathy        due to bowel preparation for colonoscopy with sodium phosphates)    -   rhabdomyolysis (breakdown of muscle tissue)—the resultant        release of myoglobin in the blood affects the kidney; it can be        caused by injury (especially crush injury and extensive blunt        trauma), statins, stimulants and some other drugs    -   hemolysis (breakdown of red blood cells)—the hemoglobin damages        the tubules; it may be caused by various conditions such as        sickle-cell disease, and lupus erythematosus    -   multiple myeloma, either due to hypercalcemia or “cast        nephropathy” (multiple myeloma can also cause chronic renal        failure by a different mechanism)    -   acute glomerulonephritis which may be due to a variety of        causes, such as anti glomerular basement membrane        disease/Goodpasture's syndrome, Wegener's granulomatosis or        acute lupus nephritis with systemic lupus erythematosus

Post-renal (obstructive causes in the urinary tract) due to:

-   -   medication interfering with normal bladder emptying (e.g.        anticholinergics).    -   benign prostatic hypertrophy or prostate cancer.    -   kidney stones.    -   due to abdominal malignancy (e.g. ovarian cancer, colorectal        cancer)    -   obstructed urinary catheter.    -   drugs that can cause crystalluria and drugs that can lead to        myoglobinuria and cystitis

According to the state of the art, renal failure is diagnosed wheneither creatinine or blood urea nitrogen tests are markedly elevated inan ill patient, especially when oliguria is present. Previousmeasurements of renal function may offer comparison, which is especiallyimportant if a patient is known to have chronic renal failure as well.If the cause is not apparent, a large amount of blood tests andexamination of a urine specimen is typically performed to elucidate thecause of acute renal failure, medical ultrasonography of the renal tractis essential to rule out obstruction of the urinary tract.

An exemplary consensus criterium for the diagnosis of AKI is at leastone of the following:

-   -   Risk: serum creatinine increased 1.5 times or urine production        of less than 0.5 ml/kg body weight for 6 hours    -   Injury: creatinine 2.0 times OR urine production less than 0.5        ml/kg for 12 h    -   Failure: creatinine 3.0 times OR creatinine more than 355 μmol/l        (with a rise of more than 44) or urine output below 0.3 ml/kg        for 24 h    -   Loss: persistent AKI or complete loss of kidney function for        more than four weeks    -   End-stage Renal Disease: complete loss of kidney function for        more than three months.

A rapid increase in serum creatinine may also be an indicator for a highAKI risk following medical treatment, e.g. an impairment in renalfunction is indicated by an increase in serum creatinine by more than0.5 mg/dl or more than 25% within 3 days after medication.

Kidney biopsy may be performed in the setting of acute renal failure, toprovide a definitive diagnosis and sometimes an idea of the prognosis,unless the cause is clear and appropriate screening investigations arereassuringly negative.

To diagnose AKI, usually urine and blood tests are done and the volumeof urine produced is monitored.

The gold standard for diagnosing AKI is the measurement of serumcreatinine. Unfortunately, creatinine as marker has several limitations.On the one hand, levels of serum creatinine widely vary amongindividuals depending on age, sex, muscle mass or medication status. Onthe other hand, serum creatinine does not accurately depict kidneyfunction during acute changes in glomerular filtration as it is amarker, which can only be interpreted in steady state. Furthermorecreatinine levels do not rise until damage is severe and kidney functionalready declines. Other biomarkers such as lipocalin 2 (LCN2), alsoknown as NGAL (neutrophil gelatinase-associated lipocalin), kidneyinjury molecule 1 (KIM1), cysteine-rich angiogenic inducer 61 (CYR61),or interleukin 18 (IL18) have recently been proposed as alternativeparameters for the detection of acute kidney injury.

WO2008/017306A1 describes a diagnostic test to exclude significant renalinjury by measuring neutrophil gelatinase-associated lipocalin (NGAL).

WO2007/013919A2 describes human Gro-alpha as a marker of acute kidneyinjury.

Perco et al (European Journal of Clinical Investigation (2006) 36,753-763) describe protein biomarkers associated with acute renal failureand chronic kidney disease.

WO2004/088276A2 discloses the detection of renal tubular cell injury andrenal failure utilizing NGAL as a biomarker.

Hauser et al (Laboratory Investigation (2004) 84, 353-361) describe agene-expression pattern of donor kidney biopsies. The differences ingene-expression between kidneys from living and cadaveric organ donorsare determined. Among other genes, versican was upregulated in samplesfrom cadaveric donors as compared to samples from living donors.

US2007/0249002A1 discloses systems and methods for characterizing kidneydiseases by detection of cytokines, cytokine-related compounds andchemokines in urine, among them monocyte chemotactic protein-1 (MCP-1).

Rice et al (Renal Failure (2002) 24(6), 703-723) disclose that monocytechemoattractant protein-1 expression correlates with monocyteinfiltration in the post-ischemic kidney. MCP-1 is reportedly increasedby ischemia-reperfusion injury.

Grandaliano et al (Transplantation (1997) 63(3), 414-420) describe theMCP-1 expression and monocyte infiltration in acute renal transplantrejection.

Jee Ko et al (Nephrol Dial Transplant (2008) 23, 842-852) describeischemia/reperfusion as a major cause of acute kidney injury andquantification of inflammatory markers, among them MCP-1.

Maier et al (Shock (2000) 14(2), 187-192) describe massive chemokinetranscription, among them MCP-1, in acute renal failure due topolymicrobial sepsis.

Wang et al (J Am SocNephrol (2002) 13, 548A) describe the upregulatedchemokine gene expression, such as MCP-1, in endotoxemic acute renalfailure.

Moon et al (J Korean Med Sci (2007) 22, 810-4) associate polymorphismsin MCP-1 promoter with diabetic kidney failure.

WO2005/054502A2 discloses a method for diagnosing rejection in atransplanted subject. A list of biomarkers indicative for transplantrejection is provided.

Akalin et al (Transplantation (2001) 72, 948-953) perform a geneexpression analysis in human renal allograft biopsy samples, anddetermine gene transcripts that are upregulated during acute rejection.

Langer et al (J Am Soc Nephrol (2004) 15, 2893-2901) describe CCL19 as amarker of allograft rejection.

Yang et al (Nephrol Dial Transplant (2007) 22, 445-456) describe theassessment of tubulointerstitial injury by immunohistochemical methods,which determine several antigens, such as collagen III.

Yoshida et al (Biochemical and Biophysical Research Communications(2002) 291, 787-794) describe the gene expression in renalischemia-reperfusion. Among the genes upregulated in the AKI model wasprocollagen type III alpha 1 (Col3a1).

Forbes et al (Kidney International (2000) 57, 375-385) describe thehistology of changes in ischemic acute renal failure. Interstitialcollagen III was increased during the first few days, followed by adecrease.

Mishra et al (J Am Soc Nephrol (2003) 14, 2534-2543) describe Lipocalinas a urinary biomarker for ischemic renal injury.

WO2007/104537A2 describes methods for assessing acute transplantrejection.

Some of the gene expression products are also known to play a role inneovascularisation and inflammation:

WO2007/096142A2 describes vascular tumor markers, such as versican, anda method for identifying diseases associated with neovascularisation.

WO2005/010213A2 describes markers for detection of gastric cancer, suchas chondroitin sulphate proteoglycan 2 (CSPG2).

WO2005/024603A2 describes a method for detecting, diagnosing andtreating human renal cell carcinoma. Differential gene expressions innormal renal epithelial cells and renal cell carcinomas are identified.Among others, neuropilin 1 is determined to be differentially expressed.

Latil et al (Int. J. Cancer (Pred. Oncol.) (2000) 89, 167-171) discloseNeuropilin-1 overexpression in metastatic tumors.

Kreuter et al (Leukemia (2006) 20, 1950-1954) describe the correlationof neuropilin-1 overexpression to survival in acute myeloid leukemia.

WO99/55855A2 describes neuropilin antisense oligonucleotides sequencesto inhibit the growth of tumor cells.

WO2007/056470A2 describes anti-NRP1 antibodies capable of inhibiting aneuropilin mediated biological activity.

WO2007/041623A2 describe methods for diagnosis in systemic inflammatoryresponse syndromes employing several markers, among them CCL19.

Krumbholz et al (Journal of Neuroimmunology (2007) 190, 72-79) disclosethe upregulation of CCL19 in neuroinflammation.

Pao et al (The Journal of Immunology (2005) 175, 3235-3245) describe therole of granzyme M in immunity to infection.

EP0913692A1 describes an immunoassay for procollagen-III-C-terminalpropeptide, using specifically binding antibodies.

Chen et al (J Mol Cell Cardial (2000) 32, 1805-1819) describe connectivetissue growth factor and TGF-beta mRNA levels that were increasedfollowing myocardial infarction, which correlated well with concomitantincreases of other markers, among them type III collagen mRNA.

Krenacs et al (Blood (2003) 101(9), 3590-3593) describe the expressionof serine protease granzyme M in lymphoma.

Bade et al (International Immunology (2005) 17(11) 1419-1428) describethe differential expression of granzymes A, K and M in human peripheralblood lymphocytes.

Sayers et al (The Journal of Immunology (2001) 166, 765-771) describethe restricted expression of granzyme M in human lymphocytes usingspecific antibodies.

Patients with normal kidney function are currently not tested for anyrenal disease biomarkers. In the absence of any functional kidneydisorder, such as urine volume reduction or creatinine level, it isassumed that there is no risk for developing AKI. However, there arepatients, who have the potential to develop AKI upon certain medicaltreatment, which could be damaging to the kidney function, such assimple radiography using a contrast medium or chemotherapy. Several riskfactors for acute renal failure have been identified so far.

High-risk patients are considered those with chronic diseases that canaffect the kidneys like diabetes, hypertension and heart disease.Pregnant patients who suffer from eclampsia, a hypertensive condition,also have a high risk for kidney damage.

Some drugs are nephrotoxic, i.e. poisonous to the kidney, and thereforedamaging to the kidneys. This includes certain antibiotics likeaminoglycosides, anti-inflammatory drugs and the contrast media used inspecific X-ray tests of the urinary tract. A need therefore exists for amarker which can be used to specifically and reproducibly detect thepresence of, or predisposition to acquiring AKI clinically leading toARF.

It is the object of the present invention to provide markers to identifypatients with early onset of AKI or predisposition for experiencing ARF.

According to the invention there is provided a method of determining therisk of acute kidney injury in a patient, by determining a kidney riskfactor (KRF) in a sample from said patient, which KRF is selected fromthe group consisting of VCAN, NRP1, CCL2, CCL19, COL3A1 and GZMM. Therisk of AKI also refers to the AKI predisposition and prognosis ofdeveloping AKI or ARF, respectively. Thus, it is understood that anindividual at risk of AKI also has a predisposition and prognosis ofdeveloping AKI and/or ARF. In particular, the risk of genuine AKI isdetermined according to the invention. It is understood that thediagnostic method according to the invention commonly is employing exvivo, in particular in vitro testing.

Preferably the method according to the invention comprises determiningthe level of said KRF, which is at least 1.2 times increased, preferablyat least 1.5 times increased, compared to a control.

In a method according to the invention, which employs the determinationof at least two of said KRF, the preferred level of each KRF is at least1.2 times increased compared to a control, to distinguish patients atrisk of AKI.

In a preferred embodiment the expression of KRF is determined in saidsample, such as the polypeptide or polynucleotide level of said KRF.

The preferred method according to the invention employs a sample, whichis selected from the group consisting of tissue or physiological fluids,such as blood, serum, plasma or urine sample. Less preferred, butpossible, is the determination of a KRF in an invasive sample, such as abiopsy sample. Further preferred samples are obtained from tissues,extracts, cell cultures, cell lysates and lavage fluid.

The condition, which can be detected with the inventive methods is inparticular a patient at risk of developing AKI, which can e.g. bedetermined by using a kidney biopsy sample and also by detecting themarkers in serum, blood, plasma or urine by comparing reference valuesof non-progressive renal disease values or from healthy subjects.

Preferably the method according to the invention is applied to apatient, who is suffering from a chronic disease, such as metabolicdisease, diabetes, hypertension or heart disease.

In another preferred embodiment the patient is tested for the riskstatus according to the invention before receiving potentiallynephrotoxic medication.

According to a preferred method, the KRF is determined by microarrayhybridization with specific probes or by PCR.

In another aspect, the invention refers to a panel of biomarkers fordetermining acute renal failure or the AKI risk, consisting of at leasttwo markers selected from the group consisting of VCAN, NRP1, CCL2,CCL19, COL3A1 and GZMM. It is therefore contemplated that one or more ofsaid biomarkers are used to manufacture a diagnostic product todetermine AKI or the AKI risk.

Thus, a set of reagents for determining the AKI risk is preferablyspecifically binding to at least two markers of the panel according tothe invention.

The preferred set according to the invention comprises reagents, whichare ligands specifically binding to said markers.

Preferably the ligands are nucleotide sequence specific oligonucleotidesor antibodies or antibody fragments. It is further preferred that thereagents are labelled.

Therefore, the present invention provides a method of detection,diagnosis, prognosis, monitoring or predisposition testing of acutekidney injury by determining the amount of a marker selected from VCAN,NRP1, CCL2, CCL19, COL3A1, GZMM or any combination thereof in a sample.For the inventive method, one of these markers can be detected, or acombination of any two, three, four, five, or six of these markers, orany combination with at least one of the markers according to theinvention with a further risk factor associated with AKI.

-   -   The inventive markers are:

1. VCAN—Versican (UniGene: Hs.643801, Hs.715773, GeneID: 1462, GenBank:AA056022/AA056070): Versican is a major extracellular chondroitinsulfate proteoglycan detected in the vessel wall, where it contributesto the formation of blood vessels. It is highly expressed by aorticendothelial cells and vascular smooth muscle cells.

2. NRP1—Neuropilin 1 (UniGene: Hs.131704, GeneID: 8829, GenBank:AA098867/AA099262): NRP1 is a membrane-bound coreceptor to a tyrosinekinase receptor for both vascular endothelial growth factor (VEGF; MIM192240) and semaphorin (see SEMA3A; MM 603961) family members, NRP1plays versatile roles in angiogenesis, axon guidance, cell survival,migration, and invasion.

3. CCL2 chemokine (C—C motif) ligand 2 (UniGene: Hs.303649, GeneID:6347, GenBank: T77817/T77816): This gene is one of several cytokinegenes clustered on the q-arm of chromosome 17. Cytokines are a family ofsecreted proteins involved in immunoregulatory and inflammatoryprocesses. The protein encoded by this gene is structurally related tothe CXC subfamily of cytokines. Members of this subfamily arecharacterized by two cysteines separated by a single amino acid. Thiscytokine displays chemotactic activity for monocytes and basophils butnot for neutrophils or eosinophils. It has been implicated in thepathogenesis of diseases characterized by monocytic infiltrates, likepsoriasis, rheumatoid arthritis and atherosclerosis. It binds tochemokine receptors CCR2 and CCR4.

4. CCL19—chemokine (C—C motif) ligand 19 (UniGene: Hs.50002, GeneID:6363, GenBank: AA680186): This gene is one of several CC cytokine genesclustered on the p-arm of chromosome 9. Cytokines are a family ofsecreted proteins involved in immunoregulatory and inflammatoryprocesses. The CC cytokines are proteins characterized by two adjacentcysteines. The cytokine encoded by this gene may play a role in normallymphocyte recirculation and homing. It also plays an important role intrafficking of T cells in thymus, and in T cell and B cell migration tosecondary lymphoid organs. It specifically binds to chemokine receptorCCR7.

5. COL3A1—collagen, type III, alpha 1 (UniGene: Hs.443625, GeneID: 1281,GenBank: AI679372): This gene encodes the pro-alpha1 chains of type IIIcollagen, a fibrillar collagen that is found in extensible connectivetissues such as skin, lung, uterus, intestine and the vascular system,frequently in association with type I collagen. Mutations in this geneare associated with Ehlers-Danlos syndrome types IV, and with aortic andarterial aneurysms. Two transcripts, resulting from the use of alternatepolyadenylation signals, have been identified for this gene.

6. GZMM—granzyme M (lymphocyte met-ase 1) (UniGene; Hs.465511, GeneID:3004, GenBank: AI124941): Human natural killer (NK) cells and activatedlymphocytes express and store a distinct subset of neutral serineproteases together with proteoglycans and other immune effectormolecules in large cytoplasmic granules. These serine proteases arecollectively termed granzymes and include 4 distinct gene products:granzyme A, granzyme B, granzyme H, and Met-ase, also known as granzymeM.

These markers, including but not limited to respective polypeptides andnucleotide sequences, such as native-sequence polypeptides, isoforms,chimeric polypeptides, any derivative, part or polymorphism (includingwithout limitation splice variant) of such biomolecules, all homologs,fragments, and precursors of the markers, and modified forms of thepolypeptides and derivatives, or nucleic acids encoding suchpolypeptides, are referred to herein as “Kidney Risk Factors (s)” (KRF).

Thus, the present invention provides a panel of biomarkers that can beused in a method for detection, diagnosis, prognosis, or monitoring theacute kidney injury (AKI), including the risk for experiencing acuterenal failure (ARF) In particular, the inventive method allows thedetermination of the predisposition for developing AKI or respectiverisk stages, e.g. to distinguish between low, medium and high riskpatients.

In a specific embodiment, the invention contemplates marker setscontaining or consisting essentially of at least two, three, four, fiveor six KRF, wherein at least one of the KRFs is selected from theinventive panel, preferably at least two, three, four, five or six ofthe KRFs according to the invention. The marker sets are preferablypolypeptide or genetic marker sets representing the KRF or respectivebinders, e.g. comprising a plurality of respective polypeptides, genesor polynucleotides.

KRF are thus preferably determined by testing for KRF polypeptides andKRF polynucleotides. In the following, KRF determination always refersto the detection and/or testing for one or more KRF polypeptides or KRFpolynucleotides. KRF determination is specifically proposed in themethod according to the invention for determining the risk fordeveloping an acute kidney disease or an acute kidney disorder, and inparticular in the detection of the risk of developing AKI within ashort, medium or long-term period, depending on medical treatment andcare. Besides determining the predisposition or risk status of apatient, the markers can be used for diagnosis, monitoring, i.e.monitoring progression or therapeutic treatment, prognosis, treatment,or classification of respective kidney disease, or as markers before orafter therapy.

Preferably those patients are tested for KRF with normal kidneyfunction, where no kidney disease is diagnosed. Normal kidney functionis defined as a glomerular filtration rate above 70 ml/min, preferablyabove 80 ml/min, more preferably above 90 ml/min and essentially noproteinuria. Other endocrine functions are of no relevance in thisproposal and thus not discussed here.

The identification of a patient's risk or predisposition is essential inthe patient population that is already classified as high-risk patients.It is thus preferred to test a patient population according to theinvention, which is already classified as risk patients, for instance,patients with risk factors of age, preexisting chronic illness,malnutrition, cancer, severe trauma, or sepsis. In particular, it isindicated to test patients suffering from metabolic disease, such asdiabetic disease, hypertension or heart or vascular disease, Typically,patients suffering from AKI are not tested for the AKI risk according tothe invention.

The inventive method can also include the step of obtaining the samplefrom a patient at risk for developing acute kidney injury, e.g. beforecontrast medium administration in the course of angiography. Thus, theterm “patients” herein always includes healthy subjects. The subjectcan, e.g., be any mammal, in particular a human, but also selected frommouse, rat, hamster, cat, dog, horse, cow, pig, etc.

Reference values for the KRF are preferably obtained from a controlgroup of patients or subjects with normal expression of said KRF, or aKRF expression, that is afflicted with kidney stress conditions, such asseptic, cancer or diabetic patients, without proteinuremia or AKI, whichrepresents the appropriate reference patient group. In a particularaspect, the control comprises material derived from a pool of samplesfrom normal patients.

Thus, the method according to the invention is specifically provided fordetermining susceptibility to kidney disease, such as AKI, bydetermining a KRF in a patient comprising:

(a) obtaining a sample from a patient,

(b) detecting or identifying in the sample a KRF, and

(c) comparing the detected amount with an amount detected for areference.

The term “detect” or “detecting” includes assaying, imaging or otherwiseestablishing the presence or absence of the target KRF encoding themarkers, subunits thereof, or combinations of reagent bound targets, andthe like, or assaying for, imaging, ascertaining, establishing, orotherwise determining one or more factual characteristics of kidneydisease or similar conditions. The term encompasses diagnostic,prognostic, and monitoring applications for a KRF.

The invention also provides a method of assessing whether a patient isat risk of AKI, comprising comparing:

(a) levels of a KRF in a sample from said patient, and

(b) normal levels of a KRF in samples of the same type obtained fromcontrol patients, wherein altered levels of the KRF relative to thecorresponding normal levels is an indication that the patient has an AKIrisk, e.g. a predisposition to kidney disease, such as AKI, inparticular where detection of a level of KRF that differs significantlyfrom the standard indicates acute kidney disease or onset of kidneydisease or increased risk for developing ARF. A significant differencebetween the levels of a KRF in a patient and the normal levels is anindication that the patient has a risk of kidney disease or apredisposition to kidney disease, such as AKI.

In a preferred embodiment, the method according to the invention forassessing whether a patient has a risk of kidney disease or apre-disposition for kidney disease, higher levels of KRF in a samplerelative to the corresponding normal levels is an indication that thepatient has kidney disease or a pre-disposition for kidney disease.

The risk of acute kidney injury is indicated if the amount of a markeror the combination of markers is increased at least 1.2 times thereference value of subjects not suffering from AKI, preferably beingsubjects from a control group or healthy subjects. Usually an increasebelow a 1.5 fold increase of an individual marker reflects a relativelylow risk; at least 1.5 fold, but below 2.0 fold increase is considered amedium risk; at least 2.0 fold increase would indicate a high-risk. Ifat least two KRFs are increased, the risk is considered to be increasedas well. Thus, at least 1.2-1.4 fold increase of each of at least twoKRFs already determines the medium to high-risk stages.

In special embodiments the amount of VCAN is at least 1.5, preferably atleast 1.6, at least 1.8, at least 2, at least 3, or at least 4 times thereference value, in particular as determined by PCR with ACTB (actinbeta) as endogenous control or as determined by microarray analysis.

In special embodiments the amount of NRP1 is at least 1.5, preferably atleast 1.6, at least 1.8, at least 2, at least 3, or at least 4 times thereference value, in particular as determined by PCR with ACTB asendogenous control or as determined by microarray analysis.

In special embodiments the amount of CCL2 is at least 1.2, preferably atleast 1.5, more preferably at least 1.6, at least 1.8, at least 2, atleast 3 or at least 4 times the reference value, in particular asdetermined by PCR with ACTB as endogenous control or as determined bymicroarray analysis.

In special embodiments the amount of CCL19 is at least 1.5, preferablyat least 1.6, at least 1.8, at least 2, at least 3, or at least 4 timesthe reference value, in particular as determined by PCR with ACTB asendogenous control or as determined by microarray analysis.

In special embodiments the amount of COL3A1 is at least 1.2, preferablyat least 1.5, more preferably at least 1.6, at least 1.8, at least 2, atleast 3 or at least 4 times the reference value, in particular asdetermined by PCR with ACTB as endogenous control or as determined bymicroarray analysis.

In special embodiments the amount of GZMM is at least 1.5, preferably atleast 1.6, at least 1.8, at least 2, at least 3, or at least 4 times thereference value, in particular as determined by PCR with ACTB asendogenous control or as determined by microarray analysis.

If more than one marker is detected, the comparison is made to eachsingle reference value for each marker in the reference itself. Theinventive prognosis method can predict whether a patient is at risk ofdeveloping acute kidney injury. The higher the fold increase, the higheris the patient's risk of AKI. An elevated KRF indicates, for example,special treatment of the patient, using appropriate medication orcontrast media. The method of the invention can thus be used to evaluatea patient before, during, and after medical treatment.

Likewise, the KRF level can be compared to a cut-off concentration andthe kidney disease development potential is determined from thecomparison; wherein concentrations of KRF above the referenceconcentrations are predictive of, e.g., correlate with, kidney diseasedevelopment in the patient.

Thus, the preferred method according to the invention comprises the stepof comparing the KRF level with a predetermined standard or cut-offvalue, which is preferably at least 50% higher than the standard, morepreferred at least 60% or 70% higher, but can also be at least 100%higher.

In aspects of the methods of the invention, the methods are non-invasivefor AKI predisposition testing, which in turn allow for diagnosis of avariety of conditions or diseases associated with acute kidney disease.In particular, the invention provides a non-invasive non-surgical methodfor detection, diagnosis, monitoring, or prediction of acute kidneydisease or onset of kidney disease in a patient comprising: obtaining asample of blood, plasma, serum, urine or saliva or a tissue sample fromthe patient; subjecting the sample to a procedure to detect one or moreKRF by comparing the levels of KRF to the levels of KRF obtained from acontrol.

The invention also contemplates a method of assessing the potential of atest compound to contribute to kidney disease or onset of kidney diseasecomprising:

(a) maintaining separate aliquots of a sample from a patient in thepresence and absence of the test compound, and

(b) comparing the levels of one or more of KRF in each of the aliquots.

This is particularly useful in monitoring the KRF level in clinicaltrials. A significant difference between the levels of a KRF in analiquot maintained in the presence of or exposed to the test compoundrelative to the aliquot maintained in the absence of the test compound,indicates that the test compound potentially contributes to kidneydisease or onset of kidney disease.

Likewise, the invention according to the invention can be employed todetermine the effect of an environmental factor on kidney diseasecomprising comparing one or more KRF associated with kidney disease oronset of kidney disease in the presence and absence of the environmentalfactor.

The inventive markers can be detected in any sample of a subjectcomprising said markers e.g. where an expression of a KRF is determinedeither as polynucleotide, e.g. as mRNA, or expressed polypeptide orprotein. The comparison with the reference value should be of the samesample type.

In preferred embodiments, determining the amount of the marker or anycombination thereof comprises determining the expression of themarker(s), preferably by determining the mRNA concentration of themarker(s). To this extent, mRNA of the sample can be isolated, ifnecessary, after adequate sample preparation steps, e.g. tissuehomogenisation, and hybridized with marker specific probes, inparticular on a microarray platform with or without amplification, orprimers for PCR-based detection methods, e.g. PCR extension labellingwith probes specific for a portion of the marker mRNA. In preferredembodiments the marker(s) or a combination thereof is (are) determinedby microarray hybridization with VCAN, NRP1, CCL2, CCL19, COL3A1, GZMMspecific probes or by PCR.

Differential expression of the polynucleotides is preferably determinedby micro-array, hybridization or by amplification of the extractedpolynucleotides. The invention contemplates a gene expression profilecomprising one or more of the KRF that is associated with AKIpredisposition. This profile provides a highly sensitive and specifictest with both high positive and negative predictive values permittingdiagnosis and prediction of the patient's risk of developing disease.

For example, the invention provides a method for determining the AKIrisk in a patient comprising

(a) contacting a sample obtained from said patient with oligonucleotidesthat hybridize to a KRF, and

(b) detecting in the sample a level of polynucleotides that hybridize tothe KRF relative to a predetermined cut-off value, and therefromdetermining the AKI risk in the subject.

Within certain preferred embodiments, the amount of polynucleotides thatare mRNA are detected via polymerase chain reaction using, for example,oligonucleotide primers that hybridize to a KRF, or complements of suchpolynucleotides. Within other embodiments, the amount of mRNA isdetected using a hybridization technique, employing oligonucleotideprobes that hybridize to a KRF, or complements thereof. When using mRNAdetection, the method may be carried out by combining isolated mRNA withreagents to convert to cDNA according to standard methods and analyzingthe products to detect the presence of KRF in the sample.

In particular aspects of the invention, the methods described hereinutilize one or more KRF placed on a micro-array so that the expressionstatus of each of the markers is assessed simultaneously. In anembodiment, the invention provides a microarray comprising a defined setof KRF genes, whose expression is significantly altered by an AKI risk.The invention further relates to the use of the microarray as aprognostic tool to predict kidney disease.

In further embodiments the amount of a marker or any combination thereofis determined by the polypeptide or protein concentration of themarker(s), e.g. with marker specific ligands, such as antibodies orspecific binding partners. The binding event can, e.g., be detected bycompetitive or non-competitive methods, including the use of labelledligand or marker specific moieties, e.g. antibodies, or labelledcompetitive moieties, including a labelled marker standard, whichcompete with marker proteins for the binding event. If the markerspecific ligand is capable of forming a complex with the marker, thecomplex formation indicates expression of the markers in the sample.

In particular, the invention relates to a method for diagnosing andmonitoring acute kidney disease in a patient by quantitating a KRF in abiological sample from the subject comprising

(a) reacting the biological sample with one or more binding agentsspecific for the KRF, e.g. an antibody that is directly or indirectlylabelled with a detectable substance, and

(b) detecting the detectable substance.

KRF levels can be determined by constructing an antibody microarray, inwhich binding sites comprise immobilized, preferably monoclonalantibodies specific to a marker. The invention also relates to kits forcarrying out the methods of the invention.

The invention further contemplates the methods, compositions, and kitsdescribed herein using additional markers associated with kidneydisease. The methods described herein may be modified by includingreagents to detect the additional markers, or polynucleotides for themarkers.

Appropriate probes, specific antibodies or methods for determining thebiomarkers are known in the art, and have been used for differentpurposes. For instance, mRNA and protein concentration of versican canbe tested with respective diagnostic tools according to WO2007/096142A2and WO2005/010213A2. NRP1 mRNA or protein concentration can be testedaccording to WO2005/024603A2. NRP1 specific oligonucleotides and NRP1specific antibodies are described in WO99/55855A2 and WO2007/056470A2,respectively. CCL2 (MCP-1) specific antibodies are described inUS2007/249002A1. Rice et al (2002, see above) describe the determinationof MCP-1 mRNA or protein using the respective tools. WO2005/054503A2discloses means to determine CCL19 mRNA or protein. Antibodies oroligonucleotides specific to COL3A1 have been described in EP0913692A1and Chen et al (2000, see above), respectively. GZMM protein andnucleotide sequence can be determined using specific antibodies and PCRprimers according to the teaching of Bade et al (2005, see above) andSayers et al (2001, see above).

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include those developed and/or usedas solid phases in solid phase binding assays. Examples of suitablesolid phases include membrane filters, cellulose-based papers, beads(including polymeric, latex and paramagnetic particles), glass, siliconwafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels,SPOCC gels, and multiple-well plates. An assay strip could be preparedby coating the antibody or a plurality of antibodies in an array onsolid support. This strip could then be dipped into the test sample andthen processed quickly through washes and detection steps to generate ameasurable signal, such as a colored spot. Antibodies or otherpolypeptides may be bound to specific zones of assay devices either byconjugating directly to an assay device surface, or by indirect binding.In an example of the later case, antibodies or other polypeptides may beimmobilized on particles or other solid supports, and that solid supportimmobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react non-specifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In exemplary embodiments, the analyte is measured using standardsandwich enzyme immunoassay techniques. A first antibody which binds theanalyte is immobilized in wells of a 96 well polystyrene microplate.Analyte standards and test samples are pipetted into the appropriatewells and any analyte present is bound by the immobilized antibody.After washing away any unbound substances, a horseradishperoxidase-conjugated second antibody which binds the analyte is addedto the wells, thereby forming sandwich complexes with the analyte (ifpresent) and the first antibody. Following a wash to remove any unboundantibody-enzyme reagent, a substrate solution comprisingtetramethylbenzidine and hydrogen peroxide is added to the wells. Colordevelops in proportion to the amount of analyte present in the sample.The color development is stopped and the intensity of the color ismeasured at 540 nm or 570 nm. An analyte concentration is assigned tothe test ample by comparison to a standard curve determined from theanalyte standards.

In a further aspect the present invention provides a set of at least twodifferent marker specific moieties, each specific for a KRF to determineat least two KRFs, wherein at least one of the KRFs is selected from thepanel according to the invention, e.g. more than two, three, four, fiveor six marker specific moieties, wherein at least two or more, such asthree, four, five or six markers selected from VCAN, NRP1, CCL2, CCL19COL3A1 or GZMM can be determined.

Preferred marker combinations can be derived from the examples and Table4 below, which are reaching area under the curve (AUC) values of atleast 0.8, preferably at least 0.85, more preferred at least 0.9, e.g.exemplarily VCAN, CCL2, and COL3A1 as well as VCAN and NRP1. Likewise,any combination of at least one KRF of the panel according to theinvention with another KRF, which brings about an AUC value as describedabove, is considered a preferred combination to determine the AKI risk.

Marker specific moieties are substances which can bind to or detect atleast one of the markers for a detection method described above and arein particular marker nucleotide sequence detecting tools or markerprotein specific antibodies, including antibody fragments, such as Fab,F(ab), F(ab)′, Fv, scFv, or single chain antibodies. The marker specificmoieties can also be selected from marker nucleotide sequence specificoligonucleotides, which specifically bind to a portion of the markersequences, e.g. mRNA or cDNA, or are complementary to such a portion inthe sense or complementary anti-sense, like cDNA complementary strand,orientation.

For easy detection the moieties are preferably labelled, such as byoptical, including fluorescence, and radioactive labels.

The present invention is further illustrated by the following figuresand examples without being limited thereto.

FIGURES

FIG. 1: Results for VCAN: Arrays: 11 ta-high vs 21 ta-low (fold-changefor AA056022/AA056070: 2.40)

FIG. 2: Results for NRP1: Arrays: 11 ta-high vs 21 ta-low (fold-changefor AA098867/AA099262: 2.16)

FIG. 3: Results for CCL2: Arrays: 11 ta-high vs 21 ta-low (fold-changefor T77817/T77816: 2.19)

FIG. 4: Results for CCL19: Arrays: 11 ta-high vs 21 ta-low (fold-changefor AA680186: 3.23)

FIG. 5: Results for COL3A1: Arrays: 11 ta-high vs 21 ta-low (fold-changefor A1679372: 2.20)

FIG. 6: Results for GZMM: Arrays: 11 ta-high vs 21 ta-low (fold-changefor A1124941: 2.18)

EXAMPLES Example 1 Patient Samples

Human renal biopsies of kidney transplant donors were collected. 82kidney biopsies were included for cDNA microarray analysis. Donor kidneybiopsies were examined pre-transplantation by a pathologist and thedegree of glomerulosclerosis (gs), arteriolosclerosis (as), interstitialfibrosis (if), interstitial inflammation (ii), tubular atrophy (tc) aswell as acute tubulus damage (ta) was assessed following asemiquantitative grading system: 0—no; 1—minor; 2—moderate; 3—severedamage. Based on the histological parameter of acute tubulus damage (ta)two groups were defined, namely those samples showing no or only minortubulus damage (n=21) and the other group of samples with severe tubulusdamage (n=11). Acute tubular damage is a histological parameter stronglycorrelated to acute kidney injury and thus was used to identify markercandidates separating samples with no or only mild damage versus sampleswith severe tubulus damage. Microarray-based gene expression profilingwas performed in those 32 patients' samples, while real-time PCRvalidation experiments were performed in 18 samples.

Example 2 RNA Isolation and Microarray Hybridization

Sample preparation followed established experimental steps as describedpreviously (Hauser et al. Lab Invest 2004, Kainz et al. Am J Transpl2004). All organs were perfused with a histidine-tryptophan-ketoglutarat(HTK) cold preservation solution at 4° C. during organ procurement.Wedge biopsy of each kidney was performed under sterile conditions atthe end of the cold ischemic time right before transplantation. Thebiopsy specimens were immediately submerged in RNAlater™ (Ambion,Austin, Tex.) and stored at 4° C. for not longer than five days.

Total RNA was isolated and purified using chloroform and trizol reagent(Invitrogen, Carlsbad, Calif.), and the RNA yield and quality waschecked with the Agilent 2100 Bioanalyzer and RNA6000 LabChip® kit(Agilent, Palo Alto, Calif.). Stratagene Universal human reference RNAwas used as reference (Stratagene, La Jolla, Calif.).

Two micrograms of isolated total RNA were amplified using the RiboAmpRNA amplification kit (Arcturus, Mountain View, Calif.). The amplifiedRNA was inspected on an ethidium bromide stained 1% agarose gel and onthe Agilent 2100 Bioanalyzer.

cDNA microarrays holding 41,409 features were obtained from the StanfordUniversity Functional Genomics core facility (batches No.: shcm, shdb,shem, sheo, sher, and shfr). A type II experimental setup was used,where each of the samples was hybridized along with a common referenceto a microarray. Stratagene Universal human reference RNA, composed oftotal RNA from 10 human cell lines, served as reference. One microgramof sample and standard Stratagene Universal human reference RNA werelabeled with CyScribe cDNA post labeling kit (Amersham PharmaciaBiotech, Buckinghamshire, UK) in a two-step procedure. Samples wereloaded onto arrays and incubated for 16 hr in a water bath at 65° C.After three washing steps, the fluorescence images of the hybridizedmicroarrays were examined using a GenePix 4100A scanner (AxonInstruments, Union City, Calif.). The GenePix Pro 4.1 software was usedto grid images and to calculate spot intensities. The arrays werenumbered according to the anonymous organ donor ID and were processed inrandom order.

Example 3 Statistical Analysis and Selection of Putative Biomarkers

Signals showing intensity signal over background values lower than 1.5in either channel were excluded and the analyses were focused on geneswith valid data in at least 80% of processed samples, leaving 24123 cDNAclones in the analysis dataset. A two-sample t-test (p<0.05) and thetwo-fold-change criterion were used to identify differentially expressedgenes (DEGs) when comparing samples with no or only mild tubular damageversus samples with severe tubular damage.

The subcellular location of DEGs was determined using data stored in theSwissProt database as well as bioinformatics prediction routines basedon the protein sequence, and secreted proteins were identified. Thesecreted DEGs showing the highest fold-change values were selected forvalidation via real-time PCR experiments.

Example 4 Validation Via Real-Time PCR

Real-time PCR was performed using the TaqMan Universal PCR Master Mix,TaqMan Gene expression assays (primers and TaqMan FAM-MGB with NFQprobes) with the ABI PRISM 7300 Sequence Detection System. The followingGene expression assays were used for the six markers CCL2, CCL19, VCAN,COL3A1, GZMM, NRP1 respectively: Hs00234140_m1, Hs00171149_m1,Hs00171642_m1, Hs00164103_m1, Hs00193417_m1, Hs00826128_m1. Allinstruments and reagents were purchased from Applied Biosystems.Relative gene expression values were evaluated with the 2-ΔΔCt methodusing ACTB (actin beta) as housekeeping gene and Stratagene Universalhuman reference RNA (Stratagene, La Jolla, Calif.) as reference RNA.This reference RNA was also used as the Standard RNA in the microarrayexperiments. qRT-PCR conditions according to the manufacture's (ABI)recommendations: 10 min 95° C., 40 cycles (15 sec 95° C., 1 min 60° C.)with fluorescence reading during annealing step.

Example 5 Microarray Analysis

22 differentially expressed transcripts mapping to 18 unique genesupregulated in the samples with severe acute tubular damage could beidentified that had protein isoforms which were secreted according toinformation stored in the SwissProt database. We selected 10 of the 18genes for quantitative real-time PCR verification experiments. Selectionof these 10 was based on fold-changes, p-values, as well as informationon gene function as derived from scientific literature. These genes areversican (VCAN), neuropilin 1 (NRP1), chemokine (C—C motif) ligand 2(CCL2), chemokine (C—C motif) ligand 19 (CCL19), collagen type III alpha1 (COL3A1), granzyme M (GZMM), apolipoprotein B (APOB), complementfactor H (CFH), ficolin 1 (FCN1), and fibrinogen-like 2 (FGL2).

TABLE 1 Results Fold-change GeneName GeneSymbol p-value (array) VersicanVCAN 0.0005 2.40 Neuropilin 1 NRP1 0.0109 2.16 chemokine (C-C motif)ligand 2 CCL2 0.0141 2.19 chemokine (C-C motif) ligand 19 CCL19 0.02753.23 collagen, type III, alpha 1 COL3A1 0.0175 2.20 granzyme M GZMM0.0150 2.18 apolipoprotein B APOB 0.0457 3.59 complement factor H CFH0.0118 2.15 ficolin 1 FCN1 0.0041 2.43 fibrinogen-like 2 FGL2 0.03132.00

Example 6 Validation Via Real-Time PCR

The upregulation of six biomarkers could be validated in rtPCRexperiments, namely VCAN, NRP1, CCL2, CCL19, COL3A1, and GZMM.

TABLE 2 Results Fold- Fold- change Gene- change (rtPCR to GeneNameSymbol p-value (array) ACTB) Versican VCAN 0.0005 2.40 2.85 Neuropilin 1NRP1 0.0109 2.16 1.8 chemokine (C-C motif) ligand 2 CCL2 0.0141 2.191.26 chemokine (C-C motif) ligand 19 CCL19 0.0275 3.23 2.55 collagen,type III, alpha 1 COL3A1 0.0175 2.20 1.28 granzyme M GZMM 0.0150 2.181.86 apolipoprotein B APOB 0.0457 3.59 −1.36 complement factor H CFH0.0118 2.15 −1.75 ficolin 1 FCN1 0.0041 2.43 −1.07 fibrinogen-like 2FGL2 0.0313 2.00 −2.06

Example 7 rtPCR Results in Proximal and Distal Tubule Cells

Renal cell suspensions were prepared from unaffected parts of tumornephrectomies. Informed consent was obtained from all patients includedin the study. Cortical tissue (approximately 0.5 cm³) was dissected byremoving the inner medulla and the outer fibrous capsule followed bymechanical homogenization using a clean scalpel. Minced sample were thenpressed through a cell dissociation sieve (SIGMA ALDRICH) andtransferred into medium M199 (Invitrogen, Carlsbad, Calif.) supplementedwith 10% foetal calf serum (Invitrogen, Carlsbad, Calif.), using theplunger of a larger syringe. The obtained suspension was then furtherpassed through a 40 μm cell strainer (BD-Biosciences) in order to obtaina nearly homogenous single cell suspension of renal tubular cells.Single cell suspension was then labelled with a PE-conjugated CD13antibody (BD-Biosciences, San Jose, Calif.) and a FITC-conjugated TammHorse Fall antibody (Cedarlanes) specific for proximal or distaltubules, respectively. Cells were washed twice with MACS-buffer(Miltenyi-Biotec) and subjected to fluorescence activated cell sortingon a FACSAria cell sorter (BD-Biosciences). Typically, proximal anddistal tubule cells ranged between 1-5% in the initial cell suspension.Cell yields after cell sorting were about 500.000 cells at puritiesof >95% for both proximal and distal tubule cells. Total RNA wasisolated and purified using trizol (Invitrogen, Carlsbad, Calif.) andchloroform (Chornczynski P et al. Anal Biochem 1987 162:156-9).

Expression profiles of the six KRFs (CCL2, CCL19, VCAN, COL3A1, GZMM,NRP1) as well as two highly expressed genes in proximal and distaltubule cells, SLC34A1 and UMOD respectively, were analyzed by real timePCR. Total RNA was used for cDNA synthesis with the High Capacity cDNAReverse Transcription Kit (Part No. 4368814). Real time PCR wasperformed using TaqMan Gene Expression Master Mix (Part No. 4369016) andTaqMan Gene expression assays (CCL2—Hs00234140_m1, CCL19—Hs00171149_m1,VCAN—Hs00171642_m1, COL3A1—Hs00164103_m1, GZMM— Hs00193417_m1,NRP1—Hs00826128_m1, SLC34A1—Hs00161828_m1, UMOD—Hs00358451_m1) on an ABI7300 Sequence Detection System. Relative gene expression values wereevaluated with the 2-ΔΔCt method using ACTB (Hs99999903_m1, beta actin)as housekeeping genes and Stratagene Universal human reference RNA(Stratagene, La Jolla, Calif.) as reference. All instruments and realtime PCR reagents were purchased by Applied Biosystems (Foster City,Calif., USA). The log 2 relative expression values of sample toStratagene Universal human reference RNA are depicted in the tablebelow. The expression of all KRFs in distal tubule cells was higher ascompared to Stratagene Universal human reference RNA. CCL19, COL3A1, andNRP1 also showed higher expression levels in proximal tubule cells ascompared to Stratagene Universal human reference RNA.

TABLE 3 The log2 relative expression values of sample to standardreference RNA of the six KDFs along with two highly abundant proteins intubuli tissue are given. Expression in distal Expression in proximalKDFs tubule cells tubule cells CCL2 2.93 −0.16 CCL19 10.93 3.56 VCAN1.28 −2.22 COL3A1 1.75 0.43 GZMM 1.23 −3.28 NRP1 1.86 2.02 SLC34A1 10.1814.42 UMOD 14.61 8.21

Example 8 p-Values for Specific Combinations

Based on gene expression data of the six KRFs under study we establishedprediction rules in order to discriminate between the binary outcomeacute tubular damage or no acute tubular damage. We assessed the abilityof the prediction rule by calculating the area under the ROC curve (AUC)using the Sommer's D statistic. The relation between the area under theROC and Sommer's D is AUC=(1+Sommer's D)/2. AUC values of 1.0 indicatecomplete discrimination of the two groups based on the marker values,whereas values of 0.5 indicate random assignment.

In this study the best single predictor of progression with an AUC valueof 0.886 is VCAN, followed by COL3A1 (AUC=0.804) and GZMM (AUC=0.794).Preferred marker combinations reaching AUC values greater than 0.9 areexemplarily VCAN, CCL2, and COL3A1, as well as VCAN and NRP1. A completelisting of AUC values of the respective markers and marker combinationsbased on gene expression data is given in the table below.

TABLE 4 Results Number Model AUC  1 VCAN 0.886  2 CCL2 0.777  3 COL3A10.804  4 GZMM 0.794  5 CCL19 0.759  6 NRP1 0.777  7 VCAN CCL2 0.892  8VCAN COL3A1 0.922  9 VCAN GZMM 0.843 10 VCAN CCL19 0.902 11 VCAN NRP11.000 12 CCL2 COL3A1 0.916 13 CCL2 GZMM 0.833 14 CCL2 CCL19 0.879 15CCL2 NRP1 0.866 16 COL3A1 GZMM 0.931 17 COL3A1 CCL19 0.819 18 COL3A1NRP1 0.822 19 GZMM CCL19 0.847 20 GZMM NRP1 0.800 21 CCL19 NRP1 0.796 22VCAN CCL2 COL3A1 0.940 23 VCAN CCL2 GZMM 0.872 24 VCAN CCL2 CCL19 0.90925 VCAN CCL2 NRP1 1.000 26 VCAN COL3A1 GZMM 0.941 27 VCAN COL3A1 CCL190.924 28 VCAN COL3A1 NRP1 1.000 29 VCAN GZMM CCL19 0.894 30 VCAN GZMMNRP1 1.000 31 VCAN CCL19 NRP1 1.000 32 CCL2 COL3A1 GZMM 0.901 33 CCL2COL3A1 CCL19 0.924 34 CCL2 COL3A1 NRP1 0.900 35 CCL2 GZMM CCL19 0.835 36CCL2 GZMM NRP1 0.800 37 CCL2 CCL19 NRP1 0.906 38 COL3A1 GZMM CCL19 0.94139 COL3A1 GZMM NRP1 0.816 40 COL3A1 CCL19 NRP1 0.843 41 GZMM CCL19 NRP10.933 42 VCAN CCL2 COL3A1 GZMM 0.921 43 VCAN CCL2 COL3A1 CCL19 0.939 44VCAN CCL2 COL3A1 NRP1 1.000 45 VCAN CCL2 GZMM CCL19 0.905 46 VCAN CCL2GZMM NRP1 1.000 47 VCAN CCL2 CCL19 NRP1 1.000 48 VCAN COL3A1 GZMM CCL190.952 49 VCAN COL3A1 GZMM NRP1 1.000 50 VCAN COL3A1 CCL19 NRP1 1.000 51VCAN GZMM CCL19 NRP1 1.000 52 CCL2 COL3A1 GZMM CCL19 0.941 53 CCL2COL3A1 GZMM NRP1 0.850 54 CCL2 COL3A1 CCL19 NRP1 0.937 55 CCL2 GZMMCCL19 NRP1 0.933 56 COL3A1 GZMM CCL19 NRP1 1.000 57 VCAN CCL2 COL3A1GZMM CCL19 0.952 58 VCAN CCL2 COL3A1 GZMM NRP1 1.000 59 VCAN CCL2 COL3A1CCL19 NRP1 1.000 60 VCAN CCL2 GZMM CCL19 NRP1 1.000 61 VCAN COL3A1 GZMMCCL19 NRP1 1.000 62 CCL2 COL3A1 GZMM CCL19 NRP1 1.000 63 VCAN CCL2COL3A1 GZMM CCL19 1.000

Example 9 Clinical Correlation

Nephrotoxic substances like antibiotics, anti-inflammatory drugs orcontrast media used in specific X-ray tests may lead to acute kidneyinjury. One of these substances is iodinated contrast medium in coronaryangiography. A sera sample collection of patients undergoing coronaryangiography was initiated. One sample was collected before coronaryangiography and second sample was collected 24 hours after coronaryangiography. Creatinine values were determined as well as the estimatedglomerular filtration rate according to the formula developed at theMayo Clinic in Rochester (Ann Intern Med. 2004; 141: 929-937). Accordingto the European Society of Urogenital Radiology, contrast-induced AKI isdefined as impairment in renal function indicated by an increase inserum creatinine by >0.5 mg/dl or >25% within 3 days after contrastmedium administration. Determination of KRF(s) in serum samples takenbefore contrast medium administration allow determining the correlationto the change in serum creatinine levels thus evaluating the potentialto predict AKI.

The concentration of KRF(s) protein in serum samples is measured viaELISA technology using means well-known in the art. In brief, for asandwich ELISA setup one monoclonal antibody directed against a specificKRF is adsorbed to wells of a 96 well polystyrene microplate, followedby incubation with human serum test samples and standards at variousdilutions. After a washing step to get rid of unbound substances asecond biotinylated detection antibody is added to each well followed byaddition of HRP (horseradish peroxidase)-labeled streptavidin. Finally,ABTS (2, 2′-Azino-bis-(3-ethylbenziazoline-6-sulfonic acid)) isintroduced and the absorption is recorded. The absorption intensity isproportional to the amount of KRF in the sample. Data are evaluated bycomparison to a standard, thus resulting in quantitative concentrationvalues for the specific KRF in human serum samples under investigation.

1. A method for treating a patient in need of treatment with anantibiotic, an anti-inflammatory agent, or a contrast medium,comprising: obtaining a biological sample from the patient; measuringexpression level of at least one kidney risk factor (KRF) in the sample,wherein the KRF is selected from the group consisting of VCAN, NRP1,CCL2, CCL19, COL3A1 and GZMM; detecting upregulation of the KRF in thebiological sample with respect to one or more control samples obtainedfrom subjects without acute kidney injury disease; and administering anantibiotic other than an aminoglycoside antibiotic, an anti-inflammatoryagent other than an NSAID, or a contrast medium other than an iodinatedcontrast medium to the patient when upregulation of the KRF is detectedin the biological sample.
 2. The method of claim 1, wherein expressionlevels of at least two KRFs are detected.
 3. The method of claim 1,wherein the expression level of the KRF in the biological sample is atleast 1.2 times increased compared to the control samples.
 4. The methodof claim 1, wherein the area under a ROC curve associated with the KRFin the biological sample is at least 0.8 using the Somer's D statistic.5. The method of claim 1, wherein upregulation of a polypeptide orpolynucleotide associated with the KRF is detected.
 6. The methodaccording to claim 1, wherein the biological sample is selected from thegroup consisting of tissue, blood, serum, plasma and urine.
 7. Themethod of claim 1, wherein upregulation of the KRF is detected bymicroarray hybridization with specific probes or by PCR.
 8. The methodof claim 1, wherein upregulation of the KRF is detected with nucleotidesequence specific oligonucleotides.
 9. The method of claim 8, whereinthe oligonucleotides are labeled.
 10. The method of claim 1, whereinupregulation of the KRF is detected with antibodies or antibodyfragments.
 11. The method of claim 1, wherein the patient is human. 12.A method for treating a patient having a chronic disease selected fromthe group consisting of diabetes, hypertension, and heart disease,wherein the treatment comprises administering a drug or a contrastmedium to the patient, comprising the steps of: obtaining a biologicalsample from the patient; measuring expression level of at least onekidney risk factor (KRF) in the sample, wherein the KRF is selected fromthe group consisting of VCAN, NRP1, CCL2, CCL19, COL3A1 and GZMM;detecting upregulation of the KRF in the biological sample with respectto one or more control samples obtained from subjects without acutekidney injury disease; and administering the drug or contrast medium tothe patient when upregulation of the KRF is detected in the biologicalsample, wherein the drug or contrast medium is not nephrotoxic.
 13. Themethod of claim 12, wherein the drug or contrast medium is not an NSAID,an aminoglycoside antibiotic, an iodinated contrast medium, lithium,sodium phosphate, or an anticholinergic.
 14. The method of claim 12,wherein the drug or contrast medium is not an NSAID, an aminoglycosideantibiotic, an iodinated contrast medium, lithium, sodium phosphate, oran anticholinergic.